Pseudomonas aeruginosa (Pa) is major Gram-negative pathogen accounting for 10% of all nosocomial infections, including 17% of pneumonia cases. Pa is of particular concern for patients hospitalized with severe burns, cancer or AIDS, for recipients of organ transplants and individuals with cystic fibrosis (CF). Mortality rates are alarmingly high. The steady and inexorable increase of resistance in this organism toward multiple classes of antibiotics, including fourth-generation cephalosporins, has severely limited treatment options and there is now an urgent need for new antimicrobials directed against this pathogen. We seek to address this need by targeting penicillin-binding proteins (PBPs) of Pa for drug discovery. The validity and accessibility of PBPs as drug targets is exemplified by the transformative success of -lactam antibiotics in the 20th century, but we are now entering a new era in which the clinical utility of these agents is waning. In Pa, a major mechanism of resistance against -lactams is the expression of -lactamases, which hydrolyze the antibiotic before it can reach its PBP target. Our overall goal is to restore PBPs as targets for antimicrobials by deployment of non- -lactam inhibitors that, by definition, would not be substrates for - lactamases. In this R21 application, we propose two requisite steps toward that goal: (1) Among the nine detectable in the Pa genome, identify the PBPs that are legitimate drug targets and (2) Using a fluorescence-polarization assay developed in our laboratory, conduct high-throughput screening to find novel inhibitors of these PBPs. The outcome will be a set of candidate inhibitors that can be later optimized via chemical modification to improve their potency and drug-like properties, with the longer-term goal of developing these as therapeutic agents against Pa.